This proposal is concerned with the structural basis of binding specificity of two nucleic acid helix-destabilizing proteins (HDPs), T4 phage gene 32 protein (G32P) and calf thymus UP1, the relationship between the in vitro helix-destabilizing activity of these two proteins and their established or suspected physiologic function in DNA replication, recombination and repair, and the interactions between these proteins and their cognate DNA polymerases. Unlike G32P, the involvement of the calf thymus HDP in DNA function has not been established, and one objective of this proposal is to pursue approaches toward establishing the physiologic roles of UP1. Since UP1 specifically stimulates the action of calf thymus DNA Polymerase Alpha, we plan protein-protein crosslinking and affinity chromatographic experiments to determine if these proteins form a complex with each other. Similar experiments are planned for G32P and the T4-Coded DNA Polymerase. In the case of the T4 system, it may be possible via crosslinking approaches to shed light on the putative functional domains of G32P. We plan to prepare derivatives of polynucleotides and oligonucleotides for use in affinity-labeling studies of HDPs: Our objective here is to (a) better define the mucleic acid binding site(s) within the G32P primary structure, (b) explore the molecular basis for the differing binding modes of polynucleotides and oligonucleotides toward G32P, and (c) determine if such differences exist for UP1. The (bulk) binding properties of UP1 and G32P differ both qualitatively and quantitatively. Several approaches are planned to determine, on the molecular level, what differences exist in the recognition of single-stranded structure by these two HDPs. The binding of UP1 and G32P to nucleic acid analogs with well-defined structural differences relative to unmodified substrates will be monitored.